CN101871881A - Method for detecting protein content in solution - Google Patents
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- CN101871881A CN101871881A CN200910082087A CN200910082087A CN101871881A CN 101871881 A CN101871881 A CN 101871881A CN 200910082087 A CN200910082087 A CN 200910082087A CN 200910082087 A CN200910082087 A CN 200910082087A CN 101871881 A CN101871881 A CN 101871881A
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- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a method for detecting protein content in solution. The invention comprises the following steps. The pH value of buffer solution is adjusted to under the isoelectric point of protein. Pigment indicator always carrying positive electricity in the solution is chosen. The protein to be tested and the pigment indicator are dissolved in the buffer solution. Protein molecules and the pigment indicator form competitive absorption on an optical waveguide surface. The time history of absorption spectrum is recorded continuously until the change of the absorption spectrum stops, i.e. the competitive absorption reaches a balanced status. In the process, the protein content in the solution is detected through detecting the time history of the pigment indicator for the guided wave absorption spectrum or by detecting the time history of optical waveguide output light strength. The invention avoids the fussy pretreating process of the sample to be tested, has the advantages of simple and convenient operation, high accuracy, low cost and zero pollution and can be applied to biological, chemical, medical and daily life protein detection field.
Description
Technical field
The present invention relates to a kind of method that detects protein content in solution.
Background technology
Protein is closely connected together with various forms of vital movements as one of material base of life, its various reaction in the catalysis life entity, regulates aspects such as metabolism and control of heredity information and all plays crucial effects.Therefore the detection for protein all has important use to be worth at aspects such as clinical diagnosis, food security, biological phenomena researchs.
Traditional detection method for determination of protein has a variety of, often need during operation testing sample is carried out complicated biological or chemical pre-treatment, for example: Folin-phenol reagent process (Lowry method) makes protein generate the protein copper composition with the copper effect under alkali condition, and this compound is reduced into blue compound with phosphomolybdic acid, phosphotungstic acid thereupon; Biuret method (Biuret method) is based on the peptide bond of protein and peptide and the analog structure of biuret, and they can both form the aubergine compound with copper ion in alkaline environment; Kai Shi (Kjeldahl) nitriding at first makes the organism and concentrated sulphuric acid heat altogether, organic nitrogen changes inorganic nitrogen ammonia into, ammonia becomes ammonium sulfate with effect of sulfuric acid, the latter and highly basic effect discharge ammonia, borrow steam that ammonia is steamed to acid solution, the nitrogen content that the degree that is neutralized according to this excessive acid solution is come calculation sample is known protein content again by inference; Coomassie brilliant blue method (Bradford method) then needs Coomassie brilliant blue G-250 to close by the Van der Waals bond with protein in acid solution, the detection sensitivity of said method at 0.02mg/mL between the 10mg/mL.
According in China's milk powder standard for the relevant regulations of protein detection, the method that is adopted is a Kjeldahl, in the recent period some illegal manufacturers improve the milk powder nitrogen content by interpolation nitrogen content toward milk powder in up to 66% harmful ultrapas, obtain profit, life security to the consumer causes a significant threat, and therefore directly fast and effeciently detecting protein content also has very important meaning for daily life.
Existing utilize optical waveguide disappearance ripple detect method for determination of protein mainly be by means of the specificity of protein in conjunction with carrying out the antigen-antibody pairing, some also needs fluorescence labeling.For example, people such as A.Ymeti utilized optical waveguide Young interferometer in conjunction with microfluid system the immune response between human albumin and the antibody to be carried out studying [A.Ymeti in 2005, J.S.Kanger, J.Greve, G.A.J.Besselink, P.V.Larnbeck, R.wijn, R.G.Heideman, Biosensors and Bioelectronics, 20 (2005) 1417-1421]; People such as Y.Enami utilized gfp molecule mark testing protein in 2007, used optical waveguide technique that fluorescence intensity is measured then, realized detection [Y.Enami to protein with this, T.Fukuda, S.Suye, Applied Physics Letters, 91 (2007) 203507]; People such as Sonia Grego utilized grating coupled planar optical waveguide pattern spectrum sensor research protein specific fixation phenomenon [Sonia Grego in 2008, Jonathan R.McDaniel, Brian R.Stoner, Sensors and Actuators B, 131 (2008) 347-355]; People such as Wen-Chi Tsai detected glucose coccus enterotoxin A (SEA) [Wen-Chi Tsai by surface plasma body resonant vibration (SPR) sensor being carried out the complex surfaces functionalization in 2009, Ie-Chin Li, Sensors and Actuators B, 136 (2009) 8-12].But all need in the above-mentioned known technology to carry out pre-treatment program, and these pre-treatment processes make that the whole test process complexity is loaded down with trivial details.
Summary of the invention
The object of the present invention is to provide a kind of method and device that detects protein content in solution.
For achieving the above object, the method of detection protein content in solution provided by the invention, comprise: the pH value of regulating buffer solution is under the isoelectric point of protein, select in solution the pigment indicator of positively charged all the time for use, be dissolved in testing protein and pigment indicator in the buffer solution jointly, protein molecule and pigment indicator form competitive Adsorption in light guide surface, the continuous recording absorption spectrum over time, no longer change in time up to absorption spectrum, promptly competitive Adsorption reaches equilibrium state; In this process, by survey the pigment indicator to the absorption spectra of guided wave over time situation detect Protein content in the solution; Or by survey the optical waveguide output light intensity over time situation detect Protein content in the solution.
Protein of the present invention can be in bovine serum albumin, haemoglobin, cytochrome protein, myoglobins, collagen, immunoglobulin (Ig), casein, lysozyme, the virus protein one or more.
Buffer solution of the present invention meets the following conditions:
(1) protein and pigment indicator are dissolved in buffer solution jointly, and do not react mutually between protein, pigment indicator and the buffering solution three;
(2) the pH value of buffer solution can be regulated within the specific limits, not only satisfies the isoelectric point condition of protein but also guarantee the protein non-inactivation.
The device that is used to realize said method provided by the invention mainly comprises:
One glass optical waveguide places on the optical waveguide substrate; One sample cell places the top of flat glass optical waveguide; The both sides of sample cell respectively are provided with a prism coupler respectively; On the light path of the incident light of prism coupler and emergent light, respectively be provided with condenser lens; One light source places on the light path of condenser lens of incident light; One photoelectric detector places on the light path of condenser lens of emergent light; After the light that light source sends focuses on through condenser lens, by becoming guided wave in the prism coupler input waveguide, again by prism coupler output, transfer to spectrometer again after output light focuses on by condenser lens light signal is handled after guided wave is propagated in optical waveguide.
The present invention surveys by means of extremely sensitive light guide surface analytical technology, the optical device that needs has: plane or three-dimensional optical waveguide, spectrometer or photodiode detector etc. that broadband or monochromatic source, prism-coupled (or end face coupling, optical fiber coupling) light advances/goes out, the principle of detection are protein molecule and the pigment indicator competitive adsorption in light guide surface.The detection method of this simple novelty can be applied in easily needs occasion that protein content is detected in biology, chemistry, medical science and the daily life.
Detection method of the present invention is to utilize optical waveguide disappearance ripple absorbance measuring technology to come the indirect detection protein content by surveying protein molecule and pigment indicator in the competitive Adsorption of light guide surface, simple and efficient to handle, do not consume sample, need not other any label, as long as select suitable buffer solution just can detect each proteinoid.Test experience to two kinds of typical protein has confirmed that the measurement sensitivity of the inventive method can reach the level of existing the whole bag of tricks fully, and method of the present invention and related content are not seen report.
Description of drawings
Fig. 1 is the structural representation of optical waveguide optical spectral apparatus;
Fig. 2, Fig. 3, Fig. 4, Fig. 5 utilize the methylene blue that is adsorbed onto glass surface from different buffer solution that the optical waveguide optical spectral technology records at the absorbance of wavelength 606.45 nanometers curve over time; Wherein:
The sample of Fig. 2 correspondence is the methylene blue of pH=5.89 and the mixed aqueous solution of BSA;
The sample of Fig. 3 a, b, c, d correspondence is the methylene blue of pH=2.03 and the mixed solution of variable concentrations BSA;
The sample of Fig. 4 a, b, c, d correspondence is the methylene blue of pH=3.00 and the mixed solution of variable concentrations BSA;
The sample of Fig. 5 a, b, c, d correspondence is the methylene blue of pH=4.02 and the mixed solution of variable concentrations BSA.
Fig. 6 a, b, c are respectively corresponding to the absorbance among Fig. 3, Fig. 4, Fig. 5 and drop to the relation of BSA concentration 40% used time of peak value and the sample from peak value;
Fig. 7 utilizes the methylene blue that is adsorbed onto glass surface from the buffer solution of pH=4.02 that the optical waveguide optical spectral technology records at the absorbance of wavelength 606.45 nanometers curve over time.The sample of Fig. 7 a, b, c, d correspondence is the methylene blue of pH=4.02 and the mixed solution of variable concentrations haemoglobin.
Fig. 8 is for dropping to the relation of hemoglobin concentration 40% used time of peak value and the sample corresponding to the absorbance among Fig. 7 from peak value.
Embodiment
The invention discloses a kind of method of measuring protein content in solution, and provide measurement result, confirmed the reliability and the accuracy of the new method of this cheap and simple two kinds of typical protein.
Measure method for determination of protein among the present invention and be based on protein molecule and pigment indicator on the plane or the competitive Adsorption on three-dimensional optical waveguide surface.Protein molecule is colourless at visible light wave range usually, and direct detection is difficulty relatively, and the inventive method is surveyed protein content in the solution by surveying the pigment indicator indirectly to the absorption of guided wave.Every kind of protein all has its specific isoelectric point (PI), if the pH value of solution on the isoelectric point of protein, protein molecule is electronegative in solution so, on the contrary positively charged then.Here selected pigment indicator positively charged all the time in solution, for example (molecular formula is [C to methyl blue
16H
18N
3S]
+Cl
-), (molecular formula is [C to rhodamine 6G
28H
31N
2O
3]
+Cl
-) etc.Because not modified glass optical waveguide surface shows electronegativity, if the pH value of regulator solution is under the isoelectric point of protein, protein and pigment indicator are dissolved in buffer solution jointly, then solution is injected the sample cell that is sealed in light guide surface, because protein molecule and pigment indicator be positively charged all, they form the relation of competitive Adsorption in light guide surface.Usually the protein molecule volume is bigger, absorption is slow but absorption is firm, and pigment molecular is little, absorption is fast but adsorb insecure, if survey in conjunction with spectrometer with wideband light source, the pigment indicator should be to rise rapidly in time to drop to the process of balance then again gradually for the absorption peak of disappearance ripple.For each protein, the fall off rate that different concentration is corresponding different is so by record guided wave absorption spectra over time, just can obtain the protein content in the solution indirectly.
A kind of typical application mode of the inventive method is described below in conjunction with accompanying drawing:
Fig. 1 is the structural representation of the optical waveguide spectral measurement device that is used to detect protein content in solution among the present invention.Optical waveguide is a flat glass optical waveguide 1, places on the optical waveguide substrate 3.Above flat glass optical waveguide 1, settle a sample cell 7, be used to hold mixed solution to be measured.The both sides of sample cell 7 respectively are provided with a prism coupler 2 respectively, respectively are provided with condenser lens 4 on the light path of the incident light of prism coupler and emergent light.Light source 5 is arranged on the light path of condenser lens of incident light.Photoelectric detector 6 is a spectrometer, is arranged on the light path of condenser lens of emergent light.After the light that light source sends focuses on through condenser lens, by becoming guided wave in the prism coupler input waveguide, again by prism coupler output, transfer to spectrometer again after output light focuses on by condenser lens light signal is handled after guided wave is propagated in optical waveguide.
In the device of the present invention, the light that light source sends is transferred to condenser lens by optical fiber 8, focus on the back by becoming guided wave in the prism coupler input waveguide through condenser lens, guided wave is exported by prism coupler again propagate a segment distance in optical waveguide after, after output light focuses on by condenser lens again through Optical Fiber Transmission to spectrometer, use spectrometer to receive and processing signals here.Choose the solution of pH value under the testing protein isoelectric point as buffer solution, should guarantee that buffer solution is the material that does not all have significant reaction with protein, pigment indicator, glass optical waveguide surface, and testing protein and pigment indicator all are dissolved in buffer solution, and this condition is easy to satisfy in experiment.When the mixed solution of testing protein and pigment indicator is injected sample cell, use spectrometer continuous recording absorption spectrum no longer to change in time up to absorption spectrum over time, show that competitive Adsorption reaches equilibrium state.If being chosen near the absorbance data of a certain wavelength the peak value in the absorption spectra handles, can find for its absorbance of different protein concentrations over time trend be identical, but reach the required asynchronism(-nization) of balance, and for the protein of same concentration, in the buffer solution of different pH values, it is also different to reach the required time of competitive Adsorption balance, and the inventive method that Here it is detects the foundation of protein content.
The optical waveguide of using in the experiment is that thickness is the flat glass optical waveguide of 50 μ m, the pigment indicator is a methylene blue, light source is a halogen tungsten lamp of containing visible light wave range, spectrometer writes down one time absorption spectrum every 1 second, in the repeatedly experiment of the bovine serum albumin (BSA) of testing variable concentrations, the competitive Adsorption majority can reach balance in ten minutes.Adopt the method for adding phosphoric acid in the deionized water to dispose the buffer solution of different pH values during experiment, experiment all keeps the concentration of methylene blue in the buffer solution constant all the time at every turn, is 18.8679 μ M/L.
The isoelectric point of bovine serum albumin (BSA) molecule is 4.7, for contrast and experiment, at first uses deionized water (pH=5.89) to experimentize as buffer solution, and the concentration of BSA is 0.3770 μ M/L in the solution, and methylene blue concentration as mentioned above.Fig. 2 utilizes the methylene blue that is adsorbed onto glass surface from BSA and methylene blue mixed aqueous solution that the optical waveguide optical spectral technology records at the absorbance of wavelength 606.45 nanometers curve over time.Absorbance hereinafter described all is the measured values under this wavelength.Because the pH value of deionized water is higher than the isoelectric point of BSA, does not as can be seen from Figure 2 have the competitive Adsorption phenomenon in deionized water between BSA molecule and the methylene blue.The absorbance that Fig. 3, Fig. 4, Fig. 5 correspond respectively to the methylene blue that is adsorbed onto glass surface from the BSA of pH=2.03, pH=3.00, pH=4.02 and methylene blue mixed solution is curve over time.The buffer solution of 4 kinds of different B SA concentration is tested under each pH value, and BSA concentration is respectively 0.0377 μ M/L, 0.0754 μ M/L, 0.1885 μ M/L and 0.3770 μ M/L.Curve among Fig. 3 to Fig. 5 reflects to have tangible competitive Adsorption phenomenon between BSA molecule and the methylene blue, initial methylene blue is adsorbed onto light guide surface rapidly, absorbance rises to peak value rapidly, the BSA molecule begins absorption and makes methylene blue from the light guide surface desorption afterwards, absorbance descends gradually, finally reaches balance.For the buffer solution of a certain particular B SA concentration, its pH value is lower than the isoelectric point of protein more, and the competitive Adsorption phenomenon is obvious more; For the buffer solution of a certain specific pH value, BSA concentration is high more, and the competitive Adsorption phenomenon is obvious more.
If each the bar curve among Fig. 3 to Fig. 5 is carried out data fitting, the absorbance that can draw methylene blue under every kind of situation respectively drops to 40% used time of peak value from peak value, under different situations, time that absorbance decline is used and BSA concentration are dull regular variation relation as can be seen from Figure.Fig. 6 a, b, c be respectively under pH=2.03, pH=3.00, three kinds of situations of pH=4.02 the absorbance of methylene blue drop to the 40% used time of peak value and the graph of a relation of BSA concentration from peak value.As can be seen from Figure 5 in the buffer solution of pH=4.02 when BSA concentration is 0.0377 μ M/L, the absorbance when reaching balance can't drop to 40% of peak value, so BSA concentration is the pairing time of 0.0377 μ M/L to fail to mark among Fig. 6 c.This external application haemoglobin replaces bovine serum albumin (BSA), and having tested hemoglobin concentration equally is 4 kinds of mixed solutions of 0.0377 μ M/L, 0.0754 μ M/L, 0.1885 μ M/L and 0.3770 μ M/L, has obtained same significantly experimental result.The isoelectric point of haemoglobin is 6.8, Fig. 7 is that the methylene blue absorbance is schemed over time in the buffer solution of pH=4.02, and the absorbance of methylene blue dropped to the 40% used time of peak value and the graph of a relation of hemoglobin concentration from peak value when Fig. 8 was pH=4.02.Experimental result explanation haemoglobin has the competitive Adsorption phenomenon with methylene blue equally under its isoelectric point.
Above-mentioned experimentation has confirmed that the present invention proposes, and to detect method for determination of protein based on protein molecule and pigment indicator in the competitive Adsorption of light guide surface be fast and effectively, be not higher than at protein concentration under the situation of 0.3770 μ M/L, method of the present invention has higher sensitivity.In addition, if change the light source among Fig. 15 light source of fixed wave length into, can also detect protein content by surveying the guided wave time-dependent variation in intensity.The light-guide testing device that this wavelength is fixed is cheap and simple more.Compare with traditional measurement method for determination of protein, the method that the present invention proposes is easy and simple to handle fast, measuring accuracy is high, cost is low, pollution-free, in the Detection Techniques field very big development potentiality is arranged.
Claims (9)
1. method that detects protein content in solution comprises:
The pH value of regulating buffer solution is under the isoelectric point of protein, select in solution the pigment indicator of positively charged all the time for use, be dissolved in testing protein and pigment indicator in the buffer solution jointly, protein molecule and pigment indicator form competitive Adsorption in light guide surface, the continuous recording absorption spectrum over time, no longer change in time up to absorption spectrum, promptly competitive Adsorption reaches equilibrium state;
In this process, by survey the pigment indicator to the absorption spectra of guided wave over time situation detect Protein content in the solution; Or
By survey the optical waveguide output light intensity over time situation detect Protein content in the solution;
Described buffer solution meets the following conditions:
(1) protein and pigment indicator are dissolved in buffer solution jointly, and do not react mutually between protein, pigment indicator and the buffering solution three;
(2) the pH value of buffer solution can be regulated within the specific limits, not only satisfies the isoelectric point condition of protein but also guarantee the protein non-inactivation.
2. the method for claim 1, wherein buffer solution is water-reducible phosphoric acid of deionization or the identical or close aqueous solution of other character.
3. the method for claim 1, wherein the pigment indicator is the kation pigment.
4. as claim 1 or 3 described methods, wherein, the pigment indicator is methylene blue, methyl orchid or rhodamine 6G.
5. the method for claim 1, wherein protein is in bovine serum albumin, haemoglobin, myoglobins, cytochrome protein, collagen, immunoglobulin (Ig), casein, lysozyme, the virus protein one or more.
6. device of realizing the described detection protein content in solution of claim 1 method mainly comprises:
One glass optical waveguide places on the optical waveguide substrate;
One sample cell places the top of flat glass optical waveguide, and contacts with optical waveguide;
The both sides of sample cell respectively are provided with a prism coupler, and prism coupler contacts with optical waveguide;
On the light path of the incident light of prism coupler and emergent light, respectively be provided with condenser lens;
One wideband light source or monochromatic light light source place on the light path of condenser lens of incident light;
One spectrometer or photodiode detection instrument place on the light path of condenser lens of emergent light;
After the light that light source sends focuses on through condenser lens, by becoming guided wave in the prism coupler input waveguide, after propagating, guided wave, transfers to spectrometer again after output light focuses on by condenser lens or the photodiode detection instrument is handled light signal again by prism coupler output in optical waveguide.
7. as the device of detection protein content in solution as described in the claim 6, wherein, optical waveguide is planar optical waveguide or three-dimensional optical waveguide.
8. as the device of detection protein content in solution as described in the claim 6, wherein, the light that light source sends by Optical Fiber Transmission to the condenser lens of incident light.
9. as the device of detection protein content in solution as described in the claim 6, wherein, the emergent light after focusing on through the emergent light condenser lens by Optical Fiber Transmission to spectrometer or photodiode detection instrument.
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